Functional unit enabling controlled flow in a microfluidic device

ABSTRACT

A microfluidic device which comprises two or more microchannel structures (set  1 ), each of which comprises a structural unit which comprises (i) one or more inlet microconduits, and (ii) an outlet microconduit downstream said one or more inlet microconduits, and (iii) a flow path for a liquid passing through either of said inlet microconduits and said outlet microconduit. The device is characterized in that each outlet microconduit in said two or more microchannel structures is a restriction microconduit. There may also be a microcavity between the inlet microconduit(s) and the restriction microconduit in each microchannel structure. Typically common flow control is used for driving a liquid flow within the device. The innovative design is useful for creating flow with low inter-channel variation with respect to the microchannel structures of the device.

This application is a continuation of U.S. application Ser. No.10/244,867 filed Sep. 17, 2002 which is a continuation in part ofInternational Application No PCT/SE02/00537, which was filed on Mar. 19,2002; and claims priority to Swedish Application No. 0103117-8, whichwas filed on Sep. 17, 2001 and U.S. Provisional Application No.60/322,621, which was filed on Sep. 17, 2001.

BACKGROUND OF THE INVENTION

A. Field of Invention

The present invention relates to a microfluidic device which comprisestwo or more microchannel structures (e.g., set 1, set 2, set 3), each ofwhich comprises one or more inlet ports, one or more outlet ports, and astructural unit which is located between an inlet port and an outletport. The structural unit comprises one or more inlet microconduits,each of which communicates with an inlet port, and an outletmicroconduit, which communicates with an outlet port, and anmicrocavity, which is located between said inlet port and said outletport. More particularly, the structural unit starts at the inlet ends ofthe inlet microconduits and ends at the outlet end of the outletmicroconduit and includes valves and anti-wicking means that may bepresent at the end parts.

B. Related Art

Typically, microfluidic devices that comprise the above-mentionedstructural unit have not comprised any means that will secureparallelity with a low inter-channel variation in flow rate betweenindividual microchannel structures. The residence time for reactantswithin the individual microcavities and elsewhere in the microchannelstructures has typically varied in an unintended manner within widelimits. Depending on kind of reactants, for instance, this may heavilyinfluence the results obtained.

Magnus Gustavsson et al., (Gyros AB) have presented experimentscomprising parallel reaction (adsorption) in a microfluidic device(“Integrated sample preparation and MALDI MS on a microfluidic compactdisc (CD with improved sensitivity”, ASMS 2001). This presentationdescribed a MALDI MS integrated microfluidic affinity system based onadsorbing a protein digest to a reverse phase matrix and subsequentdesorption and transport of peptides to a combined outlet port/MALDI MStarget. The demands on reproducibility in binding, the control of liquidflow rate, and the residence time were low. Harrison et al., (WO0138865, University of Alberta) have described a solid phase extractionmethod in a singular microchannel structure by affinity binding underflow conditions. Eteshola et al., (Sensors and Actuators B 72 (2001)129-133), Sato et al., (Anal. Chem. 72 (2000) 1144-1147); and Mian etal., (WO 9721090, Gamera Biosciences), for instance, have describedperforming affinity reactions under non-flow conditions inmicrocavities.

The use of centrifugal force for moving liquids within microfluidicsystems has also been described for instance by Abaxis Inc (WO 9533986,WO 9506870, U.S. Pat. No. 5,472,603); Molecular devices (U.S. Pat. No.5,160,702); Gamera Biosciences/Tecan (WO 9721090, WO 9807019, WO9853311), WO 01877486, WO 0187487; Gyros AB/Amersham Pharmacia Biotech(WO 9955827, WO 9958245, WO 0025921, WO 0040750, WO 0056808, WO 0062042,WO 0102737, WO 0146465, WO 0147637, WO 0147638, WO 0154810, WO 0241997,WO 0241998, PCT/SE02/00531, PCT/SE02/00537, PCT/SE02/00538,PCT/SE02/00539 and PCT/SE02/01539. See also presentations made by GyrosAB at various scientific meetings: High-through put screening SNPscoring in microfabricated device, Nigel Tooke (September 99);Microfluidics in a rotating CD (Ekstrand et al.) MicroTAS 2000,Enschede, The Netherlands, May 14-18, 2000; SNP scoring in a disposablemicrofabricated CD device (Eckersten et al.) and SNP scoring in adisposable microfabricated CD device combined with solid phasePyrosequencing™ (Tooke et al.) Human Genome Meeting, HGM 2000,Vancouver, Canada, Apr. 9-12, 2000; and Integrated sample preparationand MALDI MS on a microfluidic compact disc (CD with improvedsensitivity (Magnus Gustavsson et al.) ASMS 2001 (spring 2001).

The publications above in the name Gyros AB or Amesham Pharmacia Biotechprimarily concerns nl-volumes and problems associated therewith whilethe other publications primarily aims at μl-volumes or larger.

BRIEF SUMMARY OF THE INVENTION

The present invention is drawn to a microfluidic device which comprisestwo or more microchannel structures (e.g., set 1, set 2, set 3), each ofwhich comprises one or more inlet ports, one or more outlet ports, and astructural unit which is located between an inlet port and an outletport. The structural unit comprises one or more inlet microconduits,each of which communicates with an inlet port, and an outletmicroconduit, which communicates with an outlet port, and anmicrocavity, which is located between said inlet port and said outletport. More particularly, the structural unit starts at the inlet ends ofthe inlet microconduits and ends at the outlet end of the outletmicroconduit and includes valves and anti-wicking means that may bepresent at the end parts.

A further embodiments is that a liquid aliquot passes through thestructural unit of the present invention via at least one of the inletmicroconduits, the microcavity and the outlet microconduit of thestructural unit.

Yet further, the microchannel structures of at least one set ofmicrochannel structures are identical in the sense that correspondingparts in the individual microchannel structures are essentiallyidentical. More particularly, the microfluidic device may also compriseone or more additional sets of identical microchannel structures thatare not identical to first set of microchannel structures.

In further embodiments of the present invention, reactions may beperformed in the microcavities or downstream, for instance in and/ordownstream the outlet microconduit.

Another embodiment of the present invention is a method for creating acontrolled liquid flow in parallel through a plurality of microchannelstructures of a microfluidic device, said method comprises the steps of:(a) providing a microfluidic device which comprises said plurality ofmicrochannel structures, each of which comprises a structural unit whichcomprises one or more inlet microconduits, an outlet microconduitdownstream said one or more inlet microconduits, and a flow path for aliquid passing through either of said inlet microconduits and saidoutlet microconduit; (b) providing a liquid aliquot in at least one ofsaid one or more inlet microconduits in each of the microchannelstructures; and (c) applying a driving force that creates a liquid flowthat transport each of said aliquot through the outlet microconduit ineach of the microchannel structures; wherein the outlet microconduitsare restriction microconduits, the liquid flow created in step (c) isunder common flow control, and the flow rate created in step (c) foreach of the structural units is adjusted to give the pressure drop inthe restriction microconduits.

The present invention recognizes that the presence of means for creatinga significant pressure drop (pressure drop means, flow restrictionmeans) in the outlet microconduit, possibly combined with pressure dropmeans in the microcavity, if present and common flow control for theliquid flow in the different microchannel structures/structural unitsare of benefit for controlling the liquid flow in the microchannelstructures/ structural units of the microfluidic device.

Significant pressure drop contemplates that the pressure drop at theselocations is larger than the total inter-channel variation in flowresistance emanating from positions upstream and downstream thelocations concerned in the structural units (primarily upstream theoutlet microconduit). The term “restriction microconduit” designatesoutlet microconduits that comply with this criterion.

Yet further, the present invention has also recognized that furtherbenefits may be accomplished if one or more of the following featuresare also complied with the inventive microfluidic device: (a) porousmatrix is placed in the microcavity or immediately downstream thismicrocavity for creating a significant pressure drop along the matrix;(b) packed bed of monosized particles instead of polysized particles inthe microcavity will lower the inter-channel variation in pressure dropalong the microcavity; (c) pulse giving increased flow will assist inovercoming inter-channel variations in flow resistance of the individualmicrochannel structures (this in particular applies when initiating flowand/or when the liquid is to pass through branchings and curvatures);and (d) an anti-wicking means in inner edges downstream and in closeproximity to the outlet end of the outlet microconduit, which leadswaste liquids from the microcavity.

The microcavity (105,205) may also contain a solid phase with animmobilized affinity reactant, which is to react with an affinitycounterpart that is present as a solute in the liquid flow. The productobtained is an immobilized affinity complex. For these variants theinventors have recognized benefits for: (e) excess of solid phaseaffinity reactant (immobilized reactant) in the reaction microcavity;and (f) selecting flow rates that accomplish residence times ≧0.010seconds for the formation of complex in the reaction microcavity. It isalso envisioned that similar effects will also be accomplished for otherimmobilized reactants and heterogeneous reactions. Residence time refersto the time it takes for a liquid aliquot to pass through themicrocavity (e.g., comprising a solid phase).

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

For a more complete understanding of the present invention, reference isnow made to the following descriptions taken in conjunction with theaccompanying drawings.

FIG. 1 illustrates the key part of a structural unit that comprises amicrocavity and means for creating a pressure drop.

FIGS. 2A and 2B illustrate a variant of a preferred microchannelstructure that has a narrow microconduit that create a significantpressure drop. This variant has been used for the model study presentedin the experimental part. FIGS. 2A and 2B are identical except that FIG.2A gives the dimensions and FIG. 2B hydrophobic surface breaks and theirdimensions.

FIG. 3 illustrates a set of microchannel structures of the same kind asused in the experimental part. The structures are linked together by acommon distribution channel and a common waste channel.

The structures shown in FIGS. 2-3 are intended for a circular disc inwhich the microchannel structures are placed around its axis of symmetrythat also can be used as a spin axis. The arrow shows the directiontowards the center of the disc. Note the arc-like configuration. FIGS.2-3 also show the dimensions in micro-meter of various parts of thestructures.

DETAILED DESCRIPTION OF THE INVENTION

A first object of the present invention is to provide a fluidic functionthat when incorporated into a set of microchannel structures of amicrofluidic device will standardize the flow rate through themicrocavity, if present, and the outlet microconduit of the microchannelstructures of the set, i.e., to control the flow rate such that theinter-channel variation in flow rate is reduced to an acceptable level.

A second object of the present invention is to provide a robustmicrofluidic system that can be used for performing a plurality ofexperiments in parallel for determining reaction variables, e.g., asdescribed in PCT/SE02/00537. Typically the determination concernsfinding the content of analytes in samples and new and/or optimalbinder-ligand combinations, and/or to grade affinity for a range ofaffinity complexes, ligands and binders, and/or to optimize processesinvolving formation or dissociation of immobilized affinity complexesunder flow conditions.

A third object of the present invention is to provide a device and amethod for creating a parallel liquid flow with a low intra-channelvariation in flow rate through a set of microchannel structures of amicrofluidic device.

A fourth object of the present invention is to provide a structural unitthat enables a microfluidic device which comprises two or more sets ofmicrochannel structures which permits parallel liquid flow with a lowinter-channel variation within the individual sets and well defineddifferences in flow rates between the sets. One set, for instance, mayrepresent a particular liquid flow rate in a particular step of anapplication carried out within the structures, while another setrepresent another different flow rate for the corresponding step. Thisobject also includes a microfluidic device comprising this kind ofstructural units.

A. Structural Unit

The first aspect of the invention thus relates to a structural unit anda microfluidic device. The structural unit comprises one or more inletmicroconduits, each of which communicates with an inlet port, and anoutlet microconduit, which communicates with an outlet port, and anmicrocavity, which is located between said inlet port and said outletport. More particularly, the structural unit starts at the inlet ends ofthe inlet microconduits and ends at the outlet end of the outletmicroconduit and includes valves and anti-wicking means that may bepresent at the end parts.

Yet further, the structural unit and also the microfluidic device arecharacterized in that there are means for creating a significantpressure drop in the outlet microconduits (105,205,305) (restrictionmicroconduit) and possibly also in the microcavities (104,204,304), ifpresent. The flow through the individual microchannel structures, inparticular the inventive structural units (as shown in FIG. 1), on amicrofluidic device is preferably under common flow control.

1. Means for Creating Pressure Drop

In addition to leveling out the above-mentioned intra-channel variationin flow resistance, these means may also create a pressure drop that islarger than the total resistance to flow upstream the locationcomprising the pressure drop means, for instance upstream themicrocavity (104,204,304) or restriction microconduit (but within thestructural unit). Typically, the microchannel structures are designedsuch that the flow resistance and inter-channel variations in flowresistance at the end of the restriction microconduits (206,306) and/orin other downstream positions are insignificant compared to the upstreampart of a structural unit.

The inter-channel variations in flow resistance and pressure drop forthe structural unit of different microchannel structures primarilydepend on (a) variations in inner surface characteristics upstream themicrocavity (104,204,304), (b) variations in the solid phase used (forinstance the packing geometry may differ), and (c) design around theoutlet end (206) and surface variations in other positions downstreamthe restriction microconduit (205).

Significant pressure drop and/or significant flow resistance inpositions upstream and downstream the restriction microconduit are onlyat hand in parts that contain liquid. This means that the pressure dropupstream the inlet microconduit(s) is negligible, for instance.

The pressure drop means in the outlet microconduit (restrictionmicroconduit) and/or in the microcavity should give an inter-channelvariation in residence time for a liquid aliquot within the intervals ofmean residence time ±90%, such as ±75% or ±50% or ±25% for essentiallyidentical microchannel structures for which the driving force for theliquid flow is essentially the same.

The appropriate flow rate through and residence time in the reactionmicrocavity depends on a number of factors which among others aredetermined by the purpose of the passage through the microcavity, forinstance kind of reaction to take place, the volume of the microcavity,possible presence and form of a the solid phase, etc. The flow rateapplied should in most cases give a residence time of ≧0.010 secondssuch as ≧0.050 sec or ≧0.1 sec with an upper limit that typically isbelow 2 hours such as below 1 hour. Illustrative flow rates are within0.01-100 nl/sec, typically 0.1-10 nl/sec. The same ranges of flow ratealso apply to the flow in the restriction microconduit but the residencetime may be different.

Guidelines for calculating the proper pressure drop means for a liquidof a particular viscosity may be obtained from the formula forHagen-Poisseuille flow (fully developed laminar flow in apipe/microconduit):Q=π·(p1−p2)·Dh ⁴/(128·η·L)

where Q is the flow, pl−p2 is the pressure drop along the microconduit,Dh is the hydraulic diameter (=4×(cross-sectional area)/(wet perimeter),η is the viscosity of the liquid and L is the length of themicroconduit. Hydraulic diameter =hydrodynamic diameter of amicroconduit in the priority application. Values obtained by applicationof this formula are only guidelines and typically requires testingbefore useful values for the pressure drop means can be accomplished.

Based on the formula the general guidelines given below are applicable.

For example, for a microcavity (104,204,304), the pressure drop meansmay be a porous material through which the liquid flow has to pass.

Yet further, for the restriction microconduit (105,205,305), thepressure drop means typically contemplates that the largestcross-sectional area of this microconduit is less than the largestcross-sectional area of the inlet microconduits(102,103,202,203,302,303) or the microcavity (104,204,304) withpreference for ≦0.25, such as ≦0.10. Preferentially, these ranges applyfor ≧10%, such as in ≧50%, of the length of a restriction microconduit,often with absolute preference for ≧90% or the whole length of therestriction microconduit. The restriction microconduit typically endswhen its cross-sectional area becomes larger than the smallestcross-sectional area of the shortest inlet microconduits(102,103,202,203,302,303) or the microcavity (104,204,304), for instance≧4 times or 10 times larger.

Other kinds of pressure drop means are also possible in the outletmicroconduits (105,205,305). For example, the inner surface of therestriction microconduit, for instance, may be rougher than the innersurfaces upstream the restriction microconduit, such as upstream themicrocavity, and/or the length of the restriction microconduit(105,205,305) may be greater, such as ≧5 times or ≧10 times, the lengthof the shortest inlet microconduit, possibly plus the length of themicrocavity.

The term “length” of a microcavity or a microconduit refers to thedistance inside the microchannel structure between the most downstreamand the most upstream position of a microcavity/microconduit, or thedifference in radial distance for these two positions. An inletmicroconduit typically stretches from the restriction microconduit, orfrom the microcavity (if present) to and also encompasses the closestvalve function, anti-wicking function or branch in the upstreamdirection, or if none of these functions are present to the closestinlet port.

The pressure drop in the restriction microconduit (105) is proportionalto its length and inversely proportional to its hydrauliccross-sectional area. An increase in length of the restrictionmicroconduit may thus compensate for an increase in its cross-sectionalarea and vice versa.

The invention is particularly adapted to liquids that have a viscositythat is within the range of 10-1000% of the viscosity of water, i. e.,10⁻⁴-10⁻² Ns/m². The liquids are typically aqueous.

2. Common Flow Control

The term “common flow control” means that when a driving force for aliquid flow is applied in one part of a microchannel structure, therewill also be applied a driving force for liquid flow in thecorresponding part of each of the other microchannel structures of thedevice. The driving force in the individual microchannel structuresderives from the same source, e.g., spinning the device if centrifugalforce is the driving force. Moreover, an increase or a decrease indriving force in one microchannel structure is paralleled with anincrease or a decrease in the other microchannel structures. The size ofthe force (and the liquid flow rate) may differ between differentmicrochannel structures for which common flow control is applied. Incentrifugal based systems, for instance, the designs of the microchannelstructures may differ and/or the microchannel structures may be placedat different radial distances.

Common flow control primarily refers to the flow through the microcavity(104,204,304). This in particular applies when a reaction is to takeplace locally in the microcavity, for instance when a solid phase thatexhibits an immobilized reactant that is to interact with a reactantwhich is present in the liquid flow passing the solid phase/microcavity.See further elsewhere in this specification. Common flow control may beless critical in other parts of the structures and/or for other steps ofa method performed in the innovative microfluidic device.

The liquid flow may be driven by distinct means that either is presentin or external to the device. Thus, liquid flow may be created byelectroosmosis, micropumps, expanding gas etc., Another alternative isto use forces such as capillary force and inertia force includinggravitational force and centrifugal force to drive the liquid, i.e.,forces that do not require any means on the microfluidic device.Capillary flow is typically not under common flow control since itdepends on local surface characteristics.

According to a preferred variant of the invention, common flow controlis accomplished by spinning a microfluidic device in which themicrochannel structures are oriented from an inward position to anoutward position in relation to an axis of symmetry (spin axis) of asubstrate comprising the device. Typically the spin axis coincides withan axis of symmetry of the device as discussed below. Common flowcontrol also includes that centrifugal force is used to create asufficient local hydrostatic pressure within a structure to drive aliquid aliquot through an outward (downward) and/or an inward (upward)bent of a microchannel structure. See for instance WO 0146465. The useof centrifugal force for driving a liquid flow has been described in thepublications in the name of Gyros, Gamera Biosciences and Abaxis thatare referenced above, and also incorporated herein by reference.

Typical spinning speeds are within the interval 50-25000 rpm, such as50-15000 rpm. The spinning speed within a given protocol may vary, forinstance comprise sequences with individual ramps of acceleration,deceleration, and constant spinning. It may be beneficial to include apulse of increased spinning at certain positions.

B. Microfluidic Device

A microfluidic device comprises at least one, two or more enclosedmicrochannel structure through which liquid flow is used for thetransport of reactants

The terms “microformat”, “microchannel” etc., contemplate that amicrochannel structure comprises one or more cavities and/or channelsthat have a depth and/or a width that is ≦103 μm, preferably ≦102 μm.The volumes of microcavities are typically ≦1000 nl (=nl-range), such as≦500 nl or ≦100 nl or ≦50 nl, but may also be larger, i.e., in theinterval 1-1000 μl, such as 1-100 μl or 1-10 μl.

Liquid aliquots used in the invention typically have volumes in therange ≦5000 nl, such as ≦1000 nl or ≦500 nl or ≦100 nl or ≦50 nl, butmay also be in other intervals, such as 1-1000 μl, or 1-100 μl or 1-10μl. Dispensed aliquots are typically sucked completely into themicrochannels by capillarity before some other driving force is appliedto transport them downstream in the microchannel structures, typicallystepwise with one step for each functional unit.

In specific embodiments, it is intended that cross-sectional areas areperpendicular to the intended flow direction.

Yet further, the present invention is primarily intended for geometricarrangements in which the microchannel structures are present in asubstrate (e.g., a microfluidic device) that has an axis of symmetrythat may be used as a spin axis. The substrate in this context may bethe microfluidic device as such or a disc holder on which a microfluidicdisc comprising the microchannel structures may be placed. Theinnovative structural unit is in the upstream direction communicatingwith a unit, which delivers liquid to the unit via the inletmicroconduit (302,303) and communicates with or comprises an inlet port.In the downstream direction, the innovative structural unit communicateswith an outlet port via the restriction microconduit (205,305). Eachmicrochannel structure is oriented either fully or partly in an outwarddirection relative to the axis of symmetry (spin axis) thereby enablingcentrifugal force to be used for driving liquid flow. Yet further, it iscontemplated that the microchannel structures and microconduits may ormay not be oriented in a plane perpendicular to the axis of symmetry(spin axis).

In centrifugal systems a “higher” or an “upper” level/position (innerposition) will be at a shorter radial distance (inner position) comparedto a “lower” level/position (outer position). Similarly, the terms “up”,“upward”, “inwards”, and “down”, “downwards”, “outwards” etc., will meantowards and from, respectively, the spin axis. This applies if nototherwise is specified. With respect to other arrangement/substrates andconventional driving forces, i.e., gravity force, externally appliedpressure, electro-osmotically driven flows etc., these terms have theirconventional meaning.

Axes of symmetry are n-numbered (Cn) and may coincide with a spin axis.n is an integer between 2 and ∞, preferably 6, 7, 8 and larger, forinstance ∞. In preferred cases microfluidic device as such may have acircular, cylindrical, spherical or conical symmetry (C∞).

The preferred devices are typically disc-shaped with sizes and formssimilar to the conventional CD-format, e.g., in the interval from 10% upto 300% of the conventional CD-radii.

FIGS. 2A-B and 3 illustrate a microchannel structure (201,301) of theinvention adapted for a heterogeneous reaction. The structure comprisesa microcavity (204,304) (reaction microcavity) in which there may be animmobilized reactant (reactant 1) that is to react with a reactant(reactant 2) that is present in a liquid flow passing through themicrocavity. As already discussed for the general innovative concept(FIG. 1), the microcavity (204,304) is connected to one or more inletmicroconduits (202,203) each of which communicates with an inlet port(208,308,310), and a restriction microconduit (205,305) with an outletend (206,306) which in turn communicates with an outlet port (216,316).

The inlet port (208,307,308) may be located at a shorter radial distance(higher level) and the outlet port (216,316) at a larger radial distance(lower level) than the microcavity (204,304). By utilizing capillaryforce and/or other non-centrifugal forces for the introduction of aliquid into a microchannel structure, inlet ports may be located at inprinciple any radial distance (level) (e.g., more remote from the axisof symmetry (spin axis) than an outlet port and/or than the microcavity(not shown). Outlet ports may be located at a shorter radial distance(higher level) than the microcavity (204,304) as illustrated by port(225,325).

If there are two or more inlet microconduits (202,203 and 302,303), theytypically merge at the start of the restriction microconduit or beforethe microcavity (204,304), if it is present. In the case the microcavity(204,304) contains a solid phase in form of particles, the trespass intothe restriction microconduit (205,305) typically is a sharp drop in thecross-sectional area that prevents the particles from passing into therestriction microconduit.

For centrifugal systems, two inlet microconduits (202,203 and 302,303)typically form a downward bent (207,307) with shanks corresponding tothe inlet microconduits (202,203 and 302,303). The restrictionmicroconduit (205,305) is connected to the lower part of the bent, forinstance as illustrated in FIGS. 2-3 via the microcavity (204,304).

An inlet microconduit (202,302) may be connected to an inlet port(208,308) via a volume-defining unit (211) that comprises a meteringmicrocavity (212,312) connected to one of the inlet microconduits(202,302), an overflow channel (213,313) that starts in a narrow conduitpart between the metering microcavity (212,312) and the inlet unit (214)and ends in a waste function, for instance comprising a common wastereservoir/channel (215,315). The waste function may have one or moreoutlet ports (216,316). The volume-defining unit (211) typicallycomprises valve functions (217,218,317,318) that are associated with theoverflow channel (213,313) and with the outlet end of the meteringmicrocavity (212,312), respectively. For centrifugal systems, thesevalve functions are typically passive and are preferably based on localchanges in surface characteristics. The valve function (218,318) is at alarger radial distance than the valve function (217,317). This meansthat the positions of these valves are selected to support that liquidin the overflow channel (213,313) is passed into the waste function at alower spinning speed than liquid in the metering microcavity (212,312)is passed into the inlet microconduit (202,303). A volume-defining unitof this kind (211) is primarily intended for liquid aliquots that are tobe introduced with high accuracy with respect to volume. This meansliquid aliquots that contain an analyte and/or any other reagent thathas to be delivered with a high accuracy. See PCT/SE02/531 (Gyros AB)and PCT/SE02/01539 (Gyros AB), which are incorporated by referenceherein.

An alternative functional unit that may be connected to one of the inletmicroconduits (203,303) is a unit for simultaneously distributing liquidaliquots to several separate microchannel structures. The unit may be inthe form of a distribution channel (219,319) that is common for severalmicrochannel structures (201,301). For centrifugal system the channelmay have alternating upper and lower parts (220,320 and 221,321,respectively) with an inlet vent (222,322) (top vent) to ambientatmosphere in each upper part (220,320) and a liquid communication witha valve function (223,323) in each lower part (221,321) to one of theinlet conduits (203,303) of a microchannel structure (201,301). The topvents (222,322) may communicate with ambient atmosphere via a commonventing channel (224,324). The distribution channel may have one or moreinlet ports (307,310) and one or more outlet ports (225,325) (only oneshown) connected to separate upper parts. Several units may be linkedtogether in series via ending upper parts as illustrated in FIG. 3. Eachof the top vents may be combined or replaced with anti-wicking means inthe lower wall of the upper part connected to the top vent concerned.This kind of distribution system typically is used when identicalliquids are to be distributed as separate aliquots to differentmicrochannel structures. Typical liquids are buffers, reagents, washingliquids, samples etc., By utilizing distribution systems in which thelower parts (221,321) differ in volume, the volume of the aliquots willdiffer between the microchannel structures. See PCT/SE02/531 (Gyros AB)and PCT/SE02/01539 (Gyros AB), which are incorporated by referenceherein.

The outlet end (206,306) of a restriction microconduit (205,305) maymouth into a microconduit (226,326) with enlarged cross-sectional areaand communicating with ambient atmosphere as is illustrated with a wastefunction in FIGS. 2-3. This enlarged microconduit may also have otherfunctions, for instance as microchamber/microcavity for controlledmixing and addition of reactants such as in microtitration, organicmicrosynthesis, etc.

In the case that a waste function is linked directly to the restrictionmicroconduit, the waste function may comprise a waste microconduit(226,326) (belonging to the microchannel structure), which in turn maymouth into a common waste microconduit/reservoir (215,315).Alternatively, the outlet end (206,306) may open directly into a commonwaste microconduit/reservoir, or into ambient atmosphere (not shown).The cross-sectional area, typically the largest cross-sectional area, ofthe waste function in the proximity of the outlet opening (206,306)should be larger than the cross-sectional area of the restrictionmicroconduit (205,305) at its outlet end (206,306), e.g., ≧4 or ≧10times larger. These ranges also apply for enlarged microconduits(microcavities) that are located to this position but have otherfunctions. The outlet end (206,306) of the restriction microconduit(205,305) typically is at the same or at a higher level than the jointbetween an inlet microconduit and the restriction microcavity, or thanthe microcavity (204,304) (if present) (preferably its top part). Theabove discussions, which are incorporated herein, also apply to acentrifugal system that utilizes passive valves at the inlet ends of theinnovative structural unit.

A waste function typically has anti-wicking means (233,235,333,335) inclose proximity to the outlet end (206,306) in one or more edgesextending from the restriction microconduit (205,305) into the wastefunction, typically in edges that have a downward direction. In closeproximity, contemplates that these anti-wicking means always are abovethe lowest part of the restriction microconduit including within theoutlet end (206,306) (not shown). In the case that the restrictionmicroconduit (205,305) is connected to a waste microconduit (226,326),there is preferably a vent (227,327,235,335) to ambient atmosphere inthe waste microconduit (226,326). This vent typically is atapproximately the same level as or at a higher level than the outlet end(106) of the restriction microconduit (105) and may contain anti-wickingmeans (235,335). Yet further, the positioning of anti-wicking means inthe waste function also applies if the enlarged microconduit (226,326)does not comprise a waste function.

The principles outlined for pressure drop in the microchannel structureshave lead to the design of the microchannel structures given FIGS. 2-3;such as, reaction microcavities (204,304) with a depth of 100 μm andwidth of 250 μm; and restriction microconduits (205,305) with a depth of10 μm, a width of 20 μm and a length of 4.56 mm.

Yet further, other parts of the microchannel structures have a depth of100 μm. For the proper balancing out of inter-channel variations in flowresistance, there are hydrophobic surface breaks (233,333 and/or235,335) and a vent (227,327) to ambient atmosphere.

The inlet ports (208,308) are preferably connected to an inletmicrocavity (228,328), which typically is narrowing inwards themicrochannel structure and has longitudinal projections (ridges) (229)in the flow direction. These ridges will facilitate quick transport of adispensed liquid aliquot into the interior of an inlet unit. The inletports also may have a non-wettable area (typically hydrophobized) (230,cross-hatched) that will direct a dispensed liquid into the inlet unit(214). The same applies also to the other inlet ports (307,310).

The microchannel structures may be equipped with anti-wicking means atselected positions in form of changes in surface characteristics thattypically local and may be related to geometric surface characteristics(231) and/or chemical surface characteristics(232,233,235)(cross-hatched area). For aqueous liquids this means thatthe change is from hydrophilic to hydrophobic (hydrophobic surfacebreaks). The inlet vents (234,334,235,335) and the passive valves(217,223,317,323) comprise anti-wicking function. See WO 9958245,Amersham Pharmacia Biotech AB), WO 0185602 Åmic AB & Gyros AB andPCT/SE02/00531, PCT/SE02/01539 (plus the corresponding US applicationfiled in parallel) which are incorporated herein by reference.

Valves are preferably passive (217,223,218,318,317,323), i.e., passageof a liquid will depend on the applied driving force and thephysicochemical match between a liquid and the inner surface at thevalve position. Thus, movable mechanical parts are needed, for example,capillary valves that are based purely on a change in geometric surfacecharacteristics (WO 9615576 (David Sarnoff Res. Inst.) and WO 9807019(Gamera) which are incorporated herein by reference. More particularly,preferred passive valves are based on a change in chemical surfacecharacteristics, e.g., non-wettable surface breaks (hydrophobic surfacebreaks), possibly combined with changes in geometric surfacecharacteristics. Other kinds of valves may also be used.

More details about inlet units, distribution units, volume-definingunits, waste conduits, anti-wicking means and valves, in particular forcentrifugal systems, are given in PCT/SE02/00531 and PCT/SE02/01539(plus the US application filed in parallel), which are incorporatedherein by reference.

Suitable microfluidic devices may be manufactured from a planarsubstrate surface comprising a plurality of uncovered microchannelstructures that in a subsequent step are covered by another planarsubstrate (lid). See WO 9116966 (Pharnacia Biotech AB) and WO 0154810(Gyros AB) which are incorporated herein by reference. At least one ofthe substrates may be transparent, e.g., the second substrate (lid).Both substrates are preferably fabricated from plastic material, e.g.,plastic polymeric material.

Different applications require different surface characteristics. e.g.,inner surfaces of the microchannel structures may requirehydrophilization for transport of aqueous liquids and the like. See forinstance WO 0056808 (Gyros AB) and WO 0147637 (Gyros AB), which areincorporated herein by reference. Typically an essential part of theinner surfaces should have water contact angles ≦90°, such as ≦40° or≦30° or ≦20° at the temperature of use, e.g., at least the surfaces oftwo or three of the inner walls enclosing a channel should comply withthis range. Surfaces in passive valves, anti-wicking means, etc., areexcluded from these general rules.

C. Microcavity

The microcavity (104,204,304) is preferably a straight microchannel thatmay be continuously widening and/or narrowing. At least a part of thewall of the microcavity may be transparent to allow for measuring ofevents taking place within the microcavity. Transparency is with respectto the principle used for measuring.

The microcavity (104,204,304) may comprise a solid phase, which may haveeither one or both of the functions: (a) carrying an immobilizedreactant for a reaction to take place within the microcavity and/or (b)providing pressure drop means in the microcavity. Different reactantsare discussed below. Additional functions for the solid phase are as aseparation medium in size exclusion separation (gel chromatography, gelelectrophoresis etc.), support medium to reduce convection and/ordiffusion (electrophoresis such as isoelectrophoresis), support mediumin affinity-based separations (often included in the function of item(a)), etc.

In the preferred variant the solid phase is a population of porous ornon-porous particles that are packed to a bed, or a porous monolith thatwholly or partly will occupy the interior of the reaction microcavity.In the case the solid phase comprises particles there should be aretaining means associated with the downstream end of the reactionmicrocavity. This means is preferably in the form of a constriction,e.g., in the form of a barrier, that prevents the particles from leavingthe microcavity. The particle diameter/size should at least be of thesame size as or larger than the smallest dimension of the opening in theconstricted part. Another kind of retaining means is magnetic particlescombined with an externally applied magnetic field.

A porous monolith may be fabricated in one piece of material or maycomprise particles that are attached to each other. Yet further, aporous monolith may have pores that are large enough to permit masstransport of a reactant that is present in a liquid flow passing throughmonolith.

As used herein, the term “porous particles” refers to particles that canbe penetrated by a particular reactant that is present in a liquid flowpassing through a packed bed of the particles. This typically means Kavvalues within the interval of 0.4-0.95 for the reactant concerned.Non-porous particles have a Kav-value below 0.4 with respect to the samereactant. The particles may be spherical or non-spherical. With respectto non-spherical particles, diameters and sizes refer to the“hydrodynamic” diameters. Yet further, the particles are preferablymonodisperse (monosized) by which is meant that the population ofparticles placed in a reaction microcavity has a size distribution withmore than 95% of the particles within the range of the mean particlesize ±5%. Populations of particles that are outside this range arepolydisperse (polysized).

The solid phase may or may not be transparent. The material in the solidphase, e.g., the particles, is typically polymeric, for instance asynthetic polymer or a biopolymer. The term biopolymer includessemi-synthetic polymers comprising a polymer chain derived from a nativebiopolymer. The solid phase is typically hydrophilic in the case theliquid flow is aqueous. In this context hydrophilic encompasses that aporous solid phase, e.g., a packed bead, will be penetrated by water.The term also indicates that the surfaces of the particles shall exposea plurality of polar functional groups in which there is a heteroatomselected amongst oxygen, sulphur, and nitrogen. Appropriate functionalgroups can be selected amongst hydroxy groups, straight eythylene oxidegroups ([—CH₂CH₂O—]_(n), n an integer >0), amino groups, carboxy groups,sulphone groups etc, with preference for those groups that areessentially neutral independent of pH, for instance within the intervalof 2-12. A hydrophobic particle may be hydrophilized, for instance byintroducing hydrophilic groups. See for instance the experimental part.The coating technique is similar to the technique presented in WO9800709 (Pharmacia Biotech AB, Arvidsson & Ekström) which isincorporated herein by reference. The solid phase may also be the innersurfaces of the microcavity, but then its main function will be toprovide support for an immobilized reactant.

The reactant that may be immobilized to the solid phase depends on theapplication to be performed in the microchannel structure. It may forinstance be a reactant participating in an organic, an inorganic, abiochemical reaction etc. The reactant may thus be a catalytic system ora part of a catalytic system, such as a catalyst as such, a cocatalyst,a cofactor, a substrate or cosubstrate to the catalyst, an inhibitor, apromotor etc., with specific emphasis to the corresponding parts ofenzymatic systems (enzyme, cocatalyst, cofactor, coenzyme, substrate,cosubstrate etc.). The term “catalytic system” also includes linkedcatalytic systems, for instance a series of systems in which the productof the first system is the substrate of the second catalytic systemetc., and whole biological cells or part of such cells.

The reactant may be a so-called affinity reactant i.e., an affinityreactant that together with its affinity counterpart (affinity pair) iscapable of forming an affinity complex held together by affinity bonds.Affinity bonds typically are based on: (a) electrostatic interaction,(b) hydrophobic interaction, ″(c) electron-donor acceptor interaction,and/or (d) bioaffinity binding. Bioaffinity binding typically is complexin nature and comprises e.g., a combination of interactions selectedamongst variations of items (a)-(c).

Thus, in the present invention, an affinity reactant may thus: (a) beelectrically charged or chargeable, i.e., contains positively chargednitrogen (e.g., primary, secondary, tertiary or quaternary ammoniumgroups, and amidinium groups) and/or negatively charged groups (e.g.,carboxylate groups, phosphate groups, phosphonate groups, sulphategroups and sulphonate groups); (b) comprise hydrocarbyl groups and otherhydrophobic groups; (c) comprise heteroatoms, possibly linked tohydrogen, and/or sp²- and/or sp³-hybridised carbon, or (d) comprise incombination variations of items (a)-(c).

A bioaffinity reactant is a member of a bioaffinity pair. Typicalbioaffinity pairs are antigen/hapten and an antibody or an antigenbinding fragment of the antibody mimetic of an antibody; complementarynucleic acids; immunoglobulin-binding protein and immunoglobulin (forinstance IgG or an Fc-part thereof and protein A or G), lectin and thecorresponding carbohydrate, biotin and (strep)avidin, etc. The term“bioaffinity pair” includes affinity pairs in which one or both of themembers are synthetic, for instance mimicking a native member of abioaffinity pair.

The term “affinity reactant” includes a reactant that is capable ofreversible covalent binding, for instance by disulfide formation.Typical such reactants exhibit a HS— or a —S—SO_(n)— group (n =0,1 or 2,free valences bind to carbon). See U.S. Pat. No. 5,887,997 (Batista),U.S. Pat. No. 4,175,073 (Axén et al.), U.S. Pat. No. 4,563,304 (Axén etal.) and U.S. Pat. No. 4,647,655 (Axén et al.) which are incorporatedherein by reference. The term “affinity reactant” also includes areactant that is capable of binding via chelate formation, i.e., areactant that exhibits a chelating group, possibly in chelate form withremaining chelating ability. Affinity reactants typically exhibit aminoacid structure including peptide structure such as poly and oligopeptidestructure, carbohydrate structure, nucleotide structure includingnucleic acid structure, and lipid structure such as steroid structure,triglyceride structure, etc.

The techniques for immobilization of a reactant may be selected amongsttechniques that are commonly known in the field. The linkage to thesolid phase may be via covalent bonds, affinity bonds (for instancebiospecific affinity bonds), physical adsorption (mainly hydrophobicinteraction), etc. Examples of biospecific affinity bonds that can beused are bonds between strepavidin and a biotinylated affinity reactant(or vice versa), between an antibody and a haptenylated affinityreactant (or vice versa), etc.

D. Other Functional Units

A microchannel structure may also comprise additional units withseparate or combined functions enabling e.g. (1) separation ofparticulate matter from a liquid aliquot introduced via an inlet port,(2) mixing of two liquid aliquots, and (3) detection of (a) a reactantthat has passed through the microcavity (104,204,304), or (b) acomponent formed in the microcavity (104,204,304). Except for unit 3,these functional units, if present, may be present upstream themicrocavity (104,204,304).

A unit for separation of particulate matter is typically positionedupstream a volume metering unit or the two units are combined in acommon unit. Preferred separation units, volume metering units andmixing units are given in PCT/SE02/00531 and PCT/SE02/01539 (includingthe US application filed in parallel) which are incorporated herein byreference. A separation unit is typically combined with or presentupstream a volume-metering unit. A mixing unit, if present, is typicallypresent downstream a volume metering step.

E. Microchannel Structures having Restriction Microconduits providingdifferent Pressure Drops

The microfluidic device may contain microchannel structures that areessentially equal. The term equal in this context means that thestructures are essentially identical except for differences in design ofthe restriction microconduits (105,205,305) in order to allow them toprovide different pressure drops.

In one variant of the innovative microfluidic devices, the microchannelstructures are grouped into sets, each of which contains restrictionmicroconduits which are designed for essentially the same pressure drop,e.g., have the same length and cross-sectional area. In other words, theintended pressure drop vary between the sets but is essentially the samewithin a set. A particular interesting variant is a microfluidic devicewhich a) utilizes spinning and centrifugal force as discussed above todrive liquid flow; b) has the restriction microconduits within a set atthe same radial distance relative to the spin axis of the device but atother radial distances if they belong to other sets; c) has therestriction microconduits designed for a lower pressure drop at ashorter radial distance than the restriction microconduits designed fora higher pressure drop; and d) has the radial distance for therestriction microconduits of different sets adjusted to provide the sameflow rate through the restriction microconduit (102,103,202,203,302,302,303) and microcavity (104,204,304) (if present) of all themicrochannel structures of the microfluidic device.

An innovative centrifugal microfluidic device may thus comprisemicrochannel structures that are grouped into sets that differ withrespect to the length and/or cross-sectional area of their restrictionmicroconduits. Each of these sets may be arranged in an annular zone orsector thereof, that is concentric with the spin axis, for instance withrestriction microconduits that are shorter and/or have a largercross-sectional area at a shorter radial distance than restrictionmicroconduits that are longer and/or have a smaller cross-sectionalarea. One advantage of this is that the same controlled flow rate andresidence time easily can be accomplished in parallel within all therestriction microconduits/microcavities (105,205,305)/(104,204,304) ofmicrochannel structures that are essentially equal in the device byproperly adapting the radial distance between the annular zones and/orbetween the sectors of such zones. This is based on the fact that thecentrifugal force (driving force) increases with an increase in radialdistance.

In a second variant of a centrifugal microfluidic device, there arerestriction microconduits, which are designed for different pressuredrops and located at the same radial distance, i.e., in an annular zoneconcentric with the spin axis, or a sector of such a zone. In otherwords the same annular zone or sector may comprise restrictionmicroconduits of different length and/or cross-sectional areas.

In a third variant, there are restriction microconduits, which aredesigned for the same pressure drop but located at different radialdistances, i.e., in different annular zones that are concentric with thespin axis, or in sectors of such zones. In the second variant, the flowrate will differ between restriction microconduits/microcavities in thesame annular zone. In the third variant, the flow rate will differbetween restriction microconduits/microcavities of different annularzones.

These two variants and also other variants that are based on properlycombining restriction microconduits designed for different pressuredrops with selected combinations of radial distances for the restrictionmicroconduits will enable performing experiments on a microfluidicdevice in parallel under different controlled flow conditions (flowrates and/or residence times).

E. Use of the microfluidic device

One of the uses of the innovative device is a method for creating acontrolled liquid flow in parallel through a plurality of microchannelstructures of a microfluidic device. The method comprises the steps ofi) providing a microfluidic device which comprises two or moremicrochannel structures (201,301) each of which is defined herein; ii)providing a liquid aliquot in one of the one or more of the inletmicroconduits (202,203,302,303) in each of the microchannel structures(201,301); iii) applying a driving force that creates a liquid flow thattransport said aliquot through the outlet microconduit (205,305),possibly via the microcavity (204,304) (if present), of each of themicrochannel structures (201,301).

Step (ii) includes among others that a larger aliquot is dispensed to acommon inlet port and portioned into individual microchannel structuresvia a distribution unit, and/or that an aliquot is dispensed directly toeach microchannel structure as illustrated in FIGS. 2 and 3.

The method is characterized in that a) the outlet microconduits(105,205,305) are restriction microconduits as defined above, b) theliquid flow created in step iii) is under common flow control; and c)the driving force is adjusted to give the flow rate that is required bythe restriction microconduits (105,205,305), i.e., a flow rate withinthe interval given above that will give the pressure drop that themicroconduits (105,205,305) are designed.

The various features of the microfluidic device discussed elsewhere inthis specification further characterize sub-aspects of the method. Thisincludes the characteristics of the liquid as such and of the liquidflow, e.g., viscosity, flow rate, residence time, inter-channelvariation. The device utilizes in preferred variants spinning andcentrifugal force for common flow control and for creating the liquidflow in step (iii).

Other uses of the device relate to applications carried out within themicrochannel structures. Synthetic, analytical, preparative etc.,applications within chemical and biological sciences such as organic andorganic chemistry, biology, medicine, diagnosis, molecular biology etcare typical examples.

In many cases the applications includes that one or more reactions arecarried out within each of the microchannel structures, for instancewith at least one reaction within the microcavity (104,204,304) orwithin a reaction microcavity in a downstream position (not shown), forinstance linked directly to the outlet end of the restrictionmicroconduit (205,305) but upstream the waste function, if present. Thereactions, e.g., within the microcavity (104,204,304), may be: (a)homogeneous, i.e., between reactants (e.g., solutes) that are passingthrough the microcavity in a liquid flow, or (b) heterogeneous, i.e.,between a reactant that is immobilized within the microcavity and areactant (e.g., a solute) that is passing through the microcavity in aliquid flow. Typically the liquid flow is controlled and under commonflow control as described elsewhere in this specification.

In specific aspects, an application may comprise a separation that mayor may not comprise a reaction, for instance adsorptions that are basedof an affinity reaction as defined herein, and size exclusionseparations and electrophoresis that do not need to involve any reactionas such.

In general the innovative microfluidic device is well adapted toapplications in which controlled mixing or controlled addition ofreagents are needed, for instance microtitration and inorganic andorganic chemical synthesis in the microformat. Microtitration andcontrolled addition of reagents may for instance take place in aseparate reaction microcavity linked directly to the outlet end of therestriction microconduit (and upstream a waste function).

An important class of reactions that can be performed in the innovativemicrofluidic device comprises formation or dissociation of an affinitycomplex. Formation comprises that an affinity reactant that isimmobilized to a solid phase (capturing reactant) is introduced into themicrocavity prior to the reaction. During the reaction a liquid flowcomprising an affinity counterpart to the immobilized reactant is passedthrough the microcavity. Dissociation comprises that an immobilized formof an affinity complex is introduced into the microcavity prior to thedesired reaction is taking place. During the reaction a liquid flowproviding conditions for dissociation is passed through the microcavity.

Typically the result of the reaction is followed as the formation of aproduct, as the formation or disappearance of an intermediate or as theconsumption of a reactant etc. Various detector systems/reactions may beused, and measurement may be carried in reaction microcavity, e.g.,microcavity (104,204,304), and/or in one or more detection microcavitiesdownstream the microcavity in which the desired reaction is takingplace. Measurements are typically made through a transparent “window”.Alternatively, a product (including intermediate) and/or a remainingpart of a reactant is transferred via an outlet port to an externalinstrument for measurement.

Important reaction and detection systems as well as importantapplications are described in PCT/SE02/0537 (Gyros AB), which isincorporated herein, that also contains experiments verifying theusefulness of the innovative microfluidic device.

Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps. Yet further, all patent applications andissued patents that are referenced herein are incorporated herein byreference.

1-22. (canceled)
 23. A method of measuring an affinity complex, whereinsaid method comprises the steps of: (i) providing a microfluidic devicecomprising a plurality of microchannel structures that are under commonflow control, each microchannel structure comprising a reactionmicrocavity; (ii) performing essentially in parallel an experiment ineach of two or more of the plurality of microchannel structures, theexperiment in these two or more microchannel structures comprisingformation of an immobilized form of the complex and retaining under flowconditions said form within the reaction microcavity; and (iii)measuring the presentation of the complex in said reaction microcavityin each of said two or more microchannel structures.
 24. The method ofclaim 23, wherein each of the microcavities of said two or moremicrochannel structures comprises a solid phase to which an affinityreactant which is capable of being incorporated into the affinitycomplex retained in step (ii) is attached.
 25. The method of claim 23,wherein (a) the microfluidic device comprises a substrate having an axisof symmetry, (b) each microchannel structure is oriented relative theaxis of symmetry with an inlet port at shorter radial distance than thereaction microcavity, and (c) the substrate is spun around its axis ofsymmetry to drive liquid within the microchannel structures.
 26. Themethod of claim 23 wherein a) the microfluidic device comprises asubstrate having an axis of symmetry, (b) each microchannel structure isoriented radially relative the axis of symmetry with the reactionmicrocavity at a larger radial distance than a substructure deliveringliquid to the reaction microcavity, and (c) the substrate is spun aroundits axis of symmetry to drive liquid within the microchannel structures.27. The method of claim 23, wherein each of the microchannel structurescomprises a flow restriction downstream the reaction microcavity, whichcreates a pressure drop that restricts the flow through the reactionmicrocavity.
 28. The method of claim 23, wherein step (iii) is performedby measuring (a) distribution of the complex in the reaction microcavityalong the flow direction, or (b) the total amount of the complex in thereaction microcavity.
 29. The method of claim 23, wherein step (iii)comprises measuring the total amount of the complex in the reactionmicrocavity.
 30. The method of claim 23, wherein each of saidexperiments comprises formation of an immobilized form of the complexwithin the reaction microcavity.
 31. The method of claim 23, whereinsaid experiment comprises dissociating under flow conditions animmobilized form of the complex which complex is included in themicrofluidic device provided in step (i).
 32. The method of claim 23,wherein step (iii) comprises determining the distribution of the complexalong the flow direction in the reaction microcavity in each of said twoor more microchannel structures.
 33. The method of claim 23 wherein a)each microchannel structure comprises a unit for the separation ofparticulate matter placed upstream of the reaction microcavity, and b)step (ii) comprises separation of particulate matter from an aliquotintroduced via an inlet port.
 34. The method of claim 33, wherein a)said separation unit is combined with a volume-metering unit or isupstream of a volume metering unit, and b) step (ii) comprisesperforming a volume-metering step simultaneously with the separationstep or upstream of the volume metering step, respectively.